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Identifying the primary site of origin of metastatic cancer is vital for guiding treatment decisions, especially for patients with cancer of unknown primary (CUP). Despite advanced diagnostic techniques, CUP remains difficult to pinpoint and is responsible for a considerable number of cancer-related fatalities. Understanding its origin is crucial for effective management and potentially improving patient outcomes. This study introduces a machine learning framework, ONCOfind-AI, that leverages transcriptome-based gene set features to enhance the accuracy of predicting the origin of metastatic cancers. We demonstrate its potential to facilitate the integration of RNA sequencing and microarray data by using gene set scores for characterization of transcriptome profiles generated from different platforms. Integrating data from different platforms resulted in improved accuracy of machine learning models for predicting cancer origins. We validated our method using external data from clinical samples collected through the Kangbuk Samsung Medical Center and Gene Expression Omnibus. The external validation results demonstrate a top-1 accuracy ranging from 0.80 to 0.86, with a top-2 accuracy of 0.90. This study highlights that incorporating biological knowledge through curated gene sets can help to merge gene expression data from different platforms, thereby enhancing the compatibility needed to develop more effective machine learning prediction models.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor with a poor prognosis that poses challenges for diagnosis using traditional tissue-based techniques. DNA methylation alterations have emerged as potential and promising biomarkers for PDAC. In this study, we aimed to assess the diagnostic potential of a novel DNA methylation assay based on epigenetic-specific peptide nucleic acid (Epi-sPNA) in both tissue and plasma samples for detecting PDAC., Materials and Methods: The study involved 46 patients with PDAC who underwent surgical resection. Epi-TOP pancreatic assay was used to detect PDAC-specific epigenetic biomarkers. The Epi-sPNA allowed accurate and rapid methylation analysis without bisulfite sample processing. Genomic DNA extracted from paired normal pancreatic and PDAC tissues was used to assess the diagnostic efficacy of epigenetic biomarkers for PDAC. Subsequent validation was conducted on cell-free DNA (cfDNA) extracted from plasma samples, with 10 individuals represented in each group: PDAC, benign pancreatic cystic neoplasm, and healthy control., Results: The combination of seven epigenetic biomarkers ( HOXA9, TWIST, WT1, RPRM, BMP3, NPTX2 , and BNC1 ) achieved 93.5% sensitivity and 96.7% specificity in discerning normal pancreatic from PDAC tissues. Plasma cfDNA, analyzed using these markers and KRAS mutations, exhibited a substantial 90.0% sensitivity, 95.0% specificity, and an overall 93.3% accuracy for discriminating PDAC. Notably, cancer antigen 19-9 and carcinoembryonic antigen both had an accuracy of 90.0%., Conclusion: Our study suggests that analyzing seven differentially methylated genes with KRAS mutations in cfDNA using the novel Epi-TOP pancreatic assay is a potential blood-based biomarker for the diagnosis of PDAC., Competing Interests: Authors CB, EJ and KK were employed by Seasun Biomaterials. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kim, Chu, Do, Lee, Kim, Yang, Kwon, Lee, Batochir, Jo, Kim and Han.)

Background: Ezetimibe, which lowers cholesterol by blocking the intestinal cholesterol transporter Niemann-Pick C1 like 1, is reported to reduce hepatic steatosis in humans and animals. Here, we demonstrate the changes in hepatic metabolites and lipids and explain the underlying mechanism of ezetimibe in hepatic steatosis., Methods: We fed Otsuka Long-Evans Tokushima Fatty (OLETF) rats a high-fat diet (60 kcal % fat) with or vehicle (control) or ezetimibe (10 mg kg -1 ) via stomach gavage for 12 weeks and performed comprehensive metabolomic and lipidomic profiling of liver tissue. We used rat liver tissues, HepG2 hepatoma cell lines, and siRNA to explore the underlying mechanism., Results: In OLETF rats on a high-fat diet, ezetimibe showed improvements in metabolic parameters and reduction in hepatic fat accumulation. The comprehensive metabolomic and lipidomic profiling revealed significant changes in phospholipids, particularly phosphatidylcholines (PC), and alterations in the fatty acyl-chain composition in hepatic PCs. Further analyses involving gene expression and triglyceride assessments in rat liver tissues, HepG2 hepatoma cell lines, and siRNA experiments unveiled that ezetimibe's mechanism involves the upregulation of key phospholipid biosynthesis genes, CTP:phosphocholine cytidylyltransferase alpha and phosphatidylethanolamine N-methyl-transferase, and the phospholipid remodeling gene lysophosphatidylcholine acyltransferase 3., Conclusion: This study demonstrate that ezetimibe improves metabolic parameters and reduces hepatic fat accumulation by influencing the composition and levels of phospholipids, specifically phosphatidylcholines, and by upregulating genes related to phospholipid biosynthesis and remodeling. These findings provide valuable insights into the molecular pathways through which ezetimibe mitigates hepatic fat accumulation, emphasizing the role of phospholipid metabolism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yang, Suh, Jung, Lee, Liu, Do, Lee and Park.)

Objective: Abnormal autophagic function and activated inflammasomes are typical features in the liver of patients with non-alcoholic steatohepatitis (NASH). Here, we explored whether gemigliptin, a dipeptidyl peptidase 4 (DPP4) inhibitor for treatment of type 2 diabetes, can induce autophagy and regulate inflammasome activation as a potential NASH treatment independent of its anti-diabetic effect., Methods: Expression analysis was performed using human liver samples obtained from 18 subjects who underwent hepatectomy. We explored the function and mechanism of gemigliptin using a methionine- and choline-deficient diet (MCD)-induced NASH mouse model and HepG2 cells cultured in MCD-mimicking medium., Results: Autophagy was suppressed by marked decreases in the expression of ULK1 and LC3II/LC3I ratio in human NAFLD/NASH patients, a NASH mouse model, and HepG2 cells cultured with MCD-mimicking media. Surprisingly, we found that the expression of p-AMPK decreased in liver tissues from patients with steatosis but was restored in NASH patients. The expression of p-AMPK in the NASH mouse model was similar to that of the control group. Hence, these results indicate that autophagy was reduced in NASH via an AMPK-independent pathway. However, gemigliptin treatment attenuated lipid accumulation, inflammation, and fibrosis in the liver of MCD diet-fed mice with restoration of ULK1 expression and autophagy induction. In vitro, gemigliptin alleviated inflammasome activation through induction of ULK1-dependent autophagy. Furthermore, gemigliptin treatment upregulated ULK1 expression and activated AMPK even after siRNA-mediated knockdown of AMPKα1/2 and ULK1, respectively., Conclusions: Collectively, these results suggest that gemigliptin ameliorated NASH via AMPK-independent, ULK1-mediated effects on autophagy., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Colorectal cancers (CRC) are classified into consensus molecular subtypes (CMS) based on gene expression profiles. The revised classification system iCMS was proposed by considering intrinsic epithelial status, microsatellite instability (MSI), and fibrosis. This study aimed to provide molecular evidence for the adenoma-carcinoma sequence concept by examining CRC and synchronous adenomas using iCMS. Epithelial CMS cell proportion was estimated using CiberSortx, an in silico cell fractionation method that included CMS cell types among the reference cell types. A random forest (RF) model estimated the posterior probabilities of CMS classes, which were compared with the CiberSortx results. Gene expression profiles of the published iCMS signature panel were retrieved from our dataset and subjected to heatmap clustering for classification. Bulk RNA sequencing data were collected from 29 adenocarcinomas and 11 adenoma samples. CiberSortx showed all CRC contained either CMS2 or CMS3 as the major epithelial cancer cell type. The RF model classified approximately half of the CRC as CMS4, whereas CMS4 was hardly detected by CiberSortx. Because they were enriched with myofibroblasts as per the CiberSortx classification, we tentatively designated them as iCMS2-F/iCMS3-F. iCMS coupled with the application of an in silico cell fractionation method can provide the molecular dissection of CRC and adenoma.

Aims: To investigate the diagnostic performance of ultrasound-guided attenuation parameter (UGAP) for the detection of hepatic steatosis in nonalcoholic fatty liver disease (NAFLD) cohorts using histopathology as the reference standard andcomparing it with that of various imaging modalities., Materials and Methods: A total of 87 subjects who underwent UGAP, controlled attenuation parameter (CAP), and magnetic resonance imaging-based proton density fat fraction (MRI-PDFF) between December, 2020 and January, 2022 were enrolled. Of these patients, 38 patients had NAFLD. The association between UGAP and clinical and imaging parameters was assessed using Pearson's or Spearman's correlations. The area under the receiver operating characteristic curve (AUROC) was used to evaluate the diagnostic performance., Results: The UGAP and MRI-logPDFF demonstrated strong positive correlations (correlation coefficient= 0.704, P <0.0001). UGAP showed excellent diagnostic performance for distinguishing steatosis grade ≥1 with an AUROC of 0.821 (95% confidence interval [CI], 0.729-0.913), which was comparable to that of MRI-PDFF (0.829, 95%CI, 0.723-0.936). The AUROCs of BUSG (B-mode ultrasonography) (0.766, 95% CI, 0.767-0.856) and CAP (0.788, 95% CI, 0.684-0.891) were slightly lower than those of UGAP. The AUROCs of UGAP, MRI-PDFF, CAP, and BUSG for detecting steatosis grade ≥2 were 0.796 (95% CI, 0.616-0.975), 0.971 (95% CI, 0.936-1.000), 0.726 (95% CI, 0.561-0.891) and 0.774 (95% CI, 0.612-0.936), respectively., Conclusion: UGAP may be a valuable potential screening tool as a first-line assessment of liver steatosis in patients with NAFLD.

High-throughput mass-spectrometry-based quantitative proteomic analysis was performed using formalin-fixed, paraffin-embedded (FFPE) biopsy samples obtained before treatment from 13 patients with locally advanced rectal cancer (LARC), who were treated with concurrent chemoradiation therapy (CCRT) followed by surgery. Patients were divided into complete responder (CR) and non-complete responder (nCR) groups. Immunohistochemical (IHC) staining of 79 independent FFPE tissue samples was performed to validate the predictive ability of proteomic biomarker candidates. A total of 3637 proteins were identified, and the expression of 498 proteins was confirmed at significantly different levels (differentially expressed proteins-DEPs) between two groups. In Gene Ontology enrichment analyses, DEPs enriched in biological processes in the CR group included proteins linked to cytoskeletal organization, immune response processes, and vesicle-associated protein transport processes, whereas DEPs in the nCR group were associated with biosynthesis, transcription, and translation processes. Dual oxidase 2 (DUOX2) was selected as the most predictive biomarker in machine learning algorithm analysis. Further IHC validation ultimately confirmed DUOX2 as a potential biomarker for predicting the response of nCR to CCRT. In conclusion, this study suggests that the treatment response to RT may be affected by the pre-treatment tumor microenvironment. DUOX2 is a potential biomarker for the early prediction of nCR after CCRT.

Background: Inflammation is emerging as a potential mechanism of cervical carcinogenesis. However, few studies have investigated the association between host inflammatory status and the natural course of cervical precursor lesion. The aim of this study was to assess the probability of LSIL regression, associated with an inflammatory biomarker, high-sensitivity C-reactive protein (hs-CRP)., Methods: In a longitudinal cohort study, female participants were examined annually or biannually using cervical cytology between 2006 and 2015. Incident LSIL cases were included in the analysis, with regression defined as at least one consecutive normal cytologic result. A total of 520 women aged 22-64 years were followed up for LSIL regression. The multivariable-adjusted hazard ratios (HRs) for LSIL regression were estimated using a parametric proportional hazards model., Results: During 827.5 person-years of follow-up, 486 out of 520 subjects (93.5%) showed LSIL regression. After adjusting several important potential confounders, a higher quartile of hs-CRP levels was significantly associated with a lower rate of regression (for quartile 4 vs quartile 1, inverse HR 1.33; 95% CI, 1.04-1.69; P for trend = 0.028)., Conclusions: The low rate of spontaneous regression recorded in women with higher hs-CRP lends support to the role of the perturbated host inflammatory status in cervical carcinogenesis, and suggests that hs-CRP level could help monitor LSIL.

Background/aim: Lymph node metastasis is an important prognostic factor in gastric cancer patients. In node-negative (N0) gastric cancer patients, additional prognostic factors are needed to reinforce TNM staging., Patients and Methods: We semi-quantitatively recorded the presence of lymphatic, venous, and perineural invasion and evaluated the possibility that they could be used as upstaging factors in N0 gastric cancer by comparing N0 gastric cancer cases with N1 cases., Results: Venous (p<0.001) and perineural (p<0.001) invasion were important factors in the relapse-free survival of N0 patients, but lymphatic invasion was not. N0 cases with venous or perineural invasion had survival curves similar to those of N1 patients. In addition, the number of invasive features (lymphatic, venous, or perineural) was an important factor in predicting poor patient survival., Conclusion: Venous and perineural invasion were significant prognostic factors in N0 gastric cancer cases. It is necessary to record lymphatic, venous, and perineural invasion separately in the pathology report, especially in cases of N0 gastric cancer., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Pokemon is an oncogenic transcription regulator that plays a critical role in cellular differentiation. Although it has been found to be overexpressed in several types of cancer involving different organs, its role in thyroid gland has yet to be reported. The objective of this study was to evaluate the expression of Pokemon in papillary thyroid carcinoma (PTC) based on clinicopathological parameters., Methods: Tissue microarray samples derived from patients with PTC or benign thyroid disease were used to evaluate Pokemon expression based on immunohistochemical analysis. Correlations of its expression with various clinicopathological parameters were then analyzed., Results: Pokemon expression was observed in 22.0% of thyroid follicular cells from the normal group, 44.0% from the group with benign thyroid diseases, and 92.1% from the group with PTC (p < .001). The intensity of Pokemon expression was markedly higher in the PTC group. Pokemon expression level and PTC tumor size showed an inverse correlation. T1a tumors showed strong expression levels of Pokemon. However, larger tumors showed weak expression (p = .006)., Conclusions: Pokemon expression is associated with tumorigenesis of PTC, with expression showing an inverse correlation with PTC tumor size. This might be related to the negative regulation of aerobic glycolysis by Pokemon.

Background & Aims: Progranulin (PGRN) is known to promote tumorigenesis and proliferation of several types of cancer cells. However, little is known about the clinicopathological features of patients with gastrointestinal stromal tumors (GISTs) with regard to PGRN expression., Methods: A retrospective analysis was performed on patients with GISTs who underwent curative surgical resection between 2007 and 2017. PGRN expression was evaluated by immunohistochemical (IHC) analysis and semi-quantitatively categorized (no expression, 0; weak, 1+; moderate, 2+; strong, 3+). Tumors with a staining intensity of 2+ or 3+ were considered high PGRN expression., Results: Fifty-four patients were analyzed; 31 patients (57%) were male. The median age at surgery was 60 years (range, 33-79), and the most common primary site was the stomach (67%). Thirty-five patients (65%) had spindle histology; 42 patients (78%) were separated as a high-risk group according to the modified National Institutes of Health (NIH) classification. High PGRN-expressing tumors were observed in 27 patients (50%), had more epithelioid/mixed histology (68% vs. 32%; p = 0.046), and KIT exon 11 mutations (76% vs. 24%; p = 0.037). Patients with high PGRN-expressing tumors had a worse recurrence-free survival (RFS) (36% of 5-year RFS) compared to those with low PGRN-expressing tumors (96%; p<0.001). Multivariate analysis showed that high PGRN expression and old age (>60 years) were independent prognostic factors for poor RFS., Conclusions: High PGRN-expressing GISTs showed more epithelioid/mixed histology and KIT exon 11 mutations. PGRN overexpression was significantly associated with poor RFS in patients with GISTs who underwent curative resection., Competing Interests: The authors have read the journal’s policy and have the following competing interests: GS is a paid employee of A&G Pharmaceutical, Inc. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Prediction of remnant tumor is important in determining subsequent treatment options for gastric cancer patients with positive resection margin (RM) after endoscopic submucosal dissection (ESD)., Methods: Based on the assumption that pathologic factors, including the length and type of involved RM, could potentially predict residual tumor, we evaluated 451 ESD specimens in patients with early gastric cancer., Results: Of 408 cases, 37 (9.1 %) showed positive RMs. RM involvement in gastric cancer ESD specimens was associated with extended or beyond ESD criteria, greater tumor size, poor differentiation, submucosal invasion, lymphovascular invasion, and upper third location. Among the 37 positive RM cases, residual tumor was present in seven (18.9 %). The presence of residual tumor was not significantly associated with any clinicopathologic parameters except for tumor size and RM status. The total length of the involved RM was the most significant factor associated with the presence of residual tumor (P < 0.008). A total length cut-off value of 6 mm yielded a sensitivity of 85.7 % and negative predictive value of 94.7 % for predicting remnant tumor at gastrectomy following ESD., Conclusions: In conclusion, when the ESD specimen exhibits positive RM, a quantitative assessment of the involved RM should be included in the pathology report, as this can help the clinician predict remnant tumor and determine appropriate future treatment., (Copyright © 2020. Published by Elsevier GmbH.)

Progranulin (PGRN) has been characterized as an autocrine growth and survival factor and is known to stimulate tumorigenesis and proliferation of several types of cancer cell. However, little is known about the prognostic role of PGRN in colorectal cancer (CRC). A retrospective analysis was performed for patients with colorectal cancer who underwent curative resection between May 2013 and June 2015. PGRN expression in tumor cells was semi-quantitatively categorized (no expression, 0; weak/focal, 1+; moderate/focal or diffuse, 2+; strong/diffuse, 3+), and high expression was considered for tumors graded ≥2+ staining intensity. A total of 109 patients (28 stage I, 32 stage II, and 49 stage III) were analyzed. Thirty-eight patients (35%) had tumors with high PGRN expression, and there was a trend of elevated pre-operative CEA and CA19-9 levels in patients with high PGRN-expressing tumors compared to those with low PGRN-expressing tumors (CEA, 49% vs. 21%; CA19-9, 21% vs. 7%). The 3-year recurrence-free survival (3Y-RFS) and overall survival rates were 83.7% (95% CI, 76.8-90.6) and 96.0% (95% CI, 92.3-99.7), respectively. Patients with high PGRN-expressing tumors had a worse rate of 3Y-RFS (66.8%) compared to those with low PGRN-expressing tumors (92.4%; p = 0.010). Multivariate analysis showed that high PGRN expression, age (>66 years), stage (III), and perineural invasion (+) were independent prognostic factors associated with poor RFS after adjusting for confounding factors including sex, MSI, tumor location, KRAS, and lympho-vascular invasion. PGRN overexpression was significantly associated with poor RFS in patients with CRC who have undergone curative resection.

The first edition of the 'Standardized Pathology Report for Colorectal Cancer,' which was developed by the Gastrointestinal Pathology Study Group (GIP) of the Korean Society of Pathologists, was published 13 years ago. Meanwhile, there have been many changes in the pathologic diagnosis of colorectal cancer (CRC), pathologic findings included in the pathology report, and immunohistochemical and molecular pathology required for the diagnosis and treatment of colorectal cancer. In order to reflect these changes, we (GIP) decided to make the second edition of the report. The purpose of this standardized pathology report is to provide a practical protocol for Korean pathologists, which could help diagnose and treat CRC patients. This report consists of "standard data elements" and "conditional data elements." Basic pathologic findings and parts necessary for prognostication of CRC patients are classified as "standard data elements," while other prognostic factors and factors related to adjuvant therapy are classified as "conditional data elements" so that each institution could select the contents according to the characteristics of the institution. The Korean version is also provided separately so that Korean pathologists can easily understand and use this report. We hope that this report will be helpful in the daily practice of CRC diagnosis.

Purpose: BioPATH is a non-interventional study evaluating the relationship of molecular biomarkers (PTEN deletion/downregulation, PIK3CA mutation, truncated HER2 receptor [p95HER2], and tumor HER2 mRNA levels) to treatment responses in Asian patients with HER2+ advanced breast cancer treated with lapatinib and other HER2-targeted agents., Materials and Methods: Female Asian HER2+ breast cancer patients (n=154) who were candidates for lapatinib-based treatment following metastasis and having an available primary tumor biopsy specimen were included. The primary endpoint was progression-free survival (PFS). Secondary endpoints were response rate, overall survival on lapatinib, correlation between biomarker status and PFS for any previous trastuzumab-based treatment, and conversion/conservation rates of the biomarker status between tissue samples collected at primary diagnosis and at recurrence/metastasis. Potential relationships between tumor mRNA levels of HER2 and response to lapatinib-based therapy were also explored., Results: p95HER2, PTEN deletion/downregulation, and PIK3CA mutation did not demonstrate any significant co-occurrence pattern and were not predictive of clinical outcomes on either lapatinib-based treatment or any previous trastuzumab-based therapy in the metastatic setting. Proportions of tumors positive for p95HER2 expression, PIK3CA mutation, and PTEN deletion/down-regulation at primary diagnosis were 32%, 31.2%, and 56.2%, respectively. Despite limited availability of paired samples, biomarker status patterns were conserved in most samples. HER2 mRNA levels were not predictive of PFS on lapatinib., Conclusion: The prevalence of p95HER2 expression, PIK3CA mutation, and PTEN deletion/downregulation at primary diagnosis were similar to previous reports. Importantly, no difference was observed in clinical outcome based on the status of these biomarkers, consistent with reports from other studies.

Background: With the recent development of molecular tests for various biomarkers, it has become even more important to prepare adequate tissue samples. However, little is known about how the effect of cold ischemia time or formalin fixation time can affect KRAS mutation detection in colorectal cancer., Methods: This study included the results of KRAS mutation tests for colorectal cancer in 401 specimens. We investigated clinicopathologic factors that may affect DNA quality of formalin-fixed, paraffin-embedded (FFPE) tissue including specimen type, cold ischemia time, and formalin fixation time and assessed the detection rate of the KRAS mutation in samples with varying DNA quality., Results: Sample DNA quality for KRAS mutation test was better in biopsy specimens, which showed markedly shorter cold ischemia time and shorter formalin fixation time compared to resection specimens. A cold ischemia time of one hour or less was associated with better sample DNA quality. But the formalin fixation time was not a significant factor when it fell within the range performed in routine pathology diagnosis. When prolonged formalin fixation was tested, we confirmed that the specimen DNA quality gradually got worse from one month to three months., Conclusions: The biopsy specimens showed better sample DNA quality for KRAS mutation test compared to resection specimens. In a routine diagnostic pathology setting, the cold ischemia time was an important factor affecting DNA quality and the formalin fixation had a wide time range for optimal DNA quality., (Copyright © 2019. Published by Elsevier GmbH.)

Background: Although the incidence of early gastric cancer (EGC) continues to rise, there have been few studies on the intra-gastric distribution and locational characteristics of EGCs. In addition, there has been no attempt to visualize the intra-gastric distribution of EGCs using a merged tumor map., Methods: We investigated the anatomic distribution of 644 cases of EGCs and analyzed the correlation between clinicopathologic findings and location by dividing areas of the stomach vertically and transversely. Merged tumor maps were generated using 310 surgically resected cases., Results: Early gastric cancer was most commonly located in the antrum (57.5%) along the lesser curvature (37.8%). The intra-gastric distributions were similar in the merged tumor maps. Vertically, cancers of the middle third were associated with younger patient age, larger tumor size, and more frequent poorly differentiated (PD) or signet ring cell histology than cancers in other sites. Submucosal invasion was most frequently observed in the upper third. When divided transversely, tumors in the anterior or posterior wall showed more frequent PD or signet ring cell histology than those along the lesser or greater curvatures., Conclusions: EGC is the most prevalent in the antrum along the lesser curvature and has characteristic locational features, including histologic type, invasion depth, patient age, and tumor size. These results will improve the endoscopic detection rate of EGC and help to determine endoscopic resectability.

Background: The purpose of this study was to compare the degree of surgical smoke or vapor and lateral thermal damage caused by two different energy-based surgical devices (ESDs) used in colpotomy during total laparoscopic hysterectomy., Methods: Patients undergoing laparoscopic hysterectomy were randomly assigned to an ultrasonic ESD group (n = 20) or monopolar ESD group (n = 20). Colpotomy was performed using the assigned ESD. The degree of surgical smoke or vapor obstructing the laparoscopic view was assessed by two independent reviewers using a 5-point Likert scale, in which a higher score indicates worse visibility. The degree of the lateral thermal damage was measured as the width from the point of instrument application to the margins of the unchanged nearby tissue using a light microscope., Results: The baseline characteristics did not statistically differ between the two groups. The degree of surgical smoke or vapor obstructing vision was 1.2 ± 0.8 points in the ultrasonic group and 3.9 ± 0.7 points in the monopolar groups (p < 0.001). The lateral thermal damage was significantly increased in the monopolar group compared to in the ultrasound group (1500 µm [1200-2500 µm] vs. 950 µm [650-1725 µm], p = 0.037)., Conclusion: Ultrasonic ESD had better laparoscopic visibility and caused less lateral thermal damage during colpotomy compared to monopolar device.

Objective: To evaluate the diagnostic performance of ultrasound and to determine which ultrasound findings are useful to differentiate appendicitis from non-appendicitis in patients who underwent ultrasound re-evaluation owing to equivocal CT features of acute appendicitis., Methods: 62 patients who underwent CT examinations for suspected appendicitis followed by ultrasound re-evaluation owing to equivocal CT findings were included. Equivocal CT findings were considered based on the presence of only one or two findings among the CT criteria, and ultrasound re-evaluation was done based on a predefined structured report form. The diagnostic performance of ultrasound and independent variables to discriminate appendicitis from non-appendicitis were assessed., Results: There were 27 patients in the appendicitis group. The overall diagnostic performance of ultrasound re-evaluation was sensitivity of 96.3%, specificity of 91.2% and accuracy of 91.9%. In terms of the performance of individual ultrasound findings, probe-induced tenderness showed the highest accuracy (86.7%) with sensitivity of 74% and specificity of 97%, followed by non-compressibility (accuracy 71.7%, sensitivity 85.2% and specificity 60.6%). The independent ultrasound findings for discriminating appendicitis were non-compressibility (p = 0.002) and increased flow on the appendiceal wall (p = 0.001)., Conclusion: Ultrasound re-evaluation can be used to improve diagnostic accuracy in cases with equivocal CT features for diagnosing appendicitis. The presence of non-compressibility and increased vascular flow on the appendix wall are useful ultrasound findings to discriminate appendicitis from non-appendicitis. Advances in knowledge: Ultrasound re-evaluation is useful to discriminate appendicitis from non-appendicitis when CT features are inconclusive.

Protein arginine methylation is involved in cellular differentiation and proliferation. Recently, aberrant expression of protein arginine methyltransferases, which are responsible for the methylation reaction, has been reported in various types of cancer. However, there is no clear evidence regarding the prognostic value of abnormal PRMT6 expression in colorectal cancer or the effect of PRMT6 regulation on CRC cells. We investigated the expression patterns of PRMT6 in patients with stage II and III CRC. We detected nuclear expression of PRMT6 in 23.7% of carcinoma samples by immunohistochemistry. Among the clinicopathological parameters, the ratio of poorly differentiated cancer cells was approximately two-fold higher in patients with PRMT6-positive disease than in those with PRMT6-negative disease ( p = 0.002). Patients with PRMT6-positive CRC had a shorter disease-free survival than those with PRMT6-negative CRC in both univariate and multivariate analyses ( p = 0.018 and p = 0.035, respectively). siRNA-mediated inhibition of PRMT6 expression in CRC cells induced p21 WAF1/CIP1 overexpression and suppressed cell growth and colony-forming ability. Concomitantly, apoptosis was induced in PRMT6-suppressed CRC cells. These data suggest that PRMT6 can serve as a biomarker for unfavorable prognosis and as a therapeutic target in CRC., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Sphingosine kinase 1 (SPHK1) has been found to be upregulated in many different types of human malignancy and plays a crucial role in cancer development and progression. However, the potential of SPHK1 to act as a predictive and prognostic biomarker in breast cancer remains to be clarified. In the present study, SPHK1 expression was evaluated in breast cancer cell lines and 224 breast cancer tissue samples using immunohistochemical staining. Compared to the normal mammary epithelial cell line MCF-10A, SPHK1 mRNA and protein expression levels increased in the breast cancer cell lines SK-BR-3, MDA-MB-231, MDA-MB-436, and MCF-7. Immunohistochemical staining revealed SPHK1 expression to be significantly increased in breast cancer tissue compared to normal breast tissue, with 85 (37.9%) of the 224 invasive ductal carcinomas (IDC) exhibiting high SPHK1 expression. High SPHK1 expression in IDC showed a significant association with higher histological grade, distant metastasis, and triple negativity, and was shown to be an independent predictor for distant metastasis development. In addition, patients with high SPHK1 expression had significantly lower progression-free survival and overall survival rates compared to those with low SPHK1 expression. Our data suggest that SPHK1 is involved in the development and progression of breast cancer and can serve as a potential predictive biomarker of distant metastasis and patient outcome., Competing Interests: None.

Background/aim: Sphingolipid metabolites are emerging as key signaling molecules in cancer. Sphingosine kinase 1 is up-regulated in many different types of human malignancies and plays a crucial role in cancer development and progression. The utility of sphingosine kinase 1 to act as a predictive biomarker in thyroid cancer remains unclear., Materials and Methods: Sphingosine kinase 1 expression was evaluated using immunohistochemical staining in 110 formalin-fixed, paraffin-embedded papillary thyroid carcinoma tissue samples., Results: Sphingosine kinase 1 expression in papillary thyroid carcinoma tissue was significantly higher than in nodular goiter (p<0.001) or normal thyroid (p<0.001) tissue. Sphingosine kinase 1 was observed in the cytoplasm of tumor cells. Thirty-four (30.9%) of 110 papillary thyroid carcinomas exhibited high sphingosine kinase 1 expression, that was significantly associated with tumor multiplicity (p=0.004), extrathyroidal extension (p=0.013), presence of lymph node metastasis (p<0.001), and number of metastatic lymph nodes (p=0.042). In addition, high sphingosine kinase 1 expression was the only independent predictor of lymph node metastasis (p<0.001)., Conclusion: Sphingosine kinase 1 is involved in papillary thyroid carcinoma development and progression and can serve as a potential biomarker predictive of lymph node metastasis., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Cytokeratin 17 (CK17), a basal/myoepithelial cell keratin, is a poor prognostic marker for cancers of organs such as the stomach, ovary, and breast as well as a useful diagnostic marker for pancreatobiliary adenocarcinoma. However, its expression pattern and prognostic significance have not been studied in gallbladder adenocarcinoma. We constructed a tissue microarray from samples from 82 consecutive patients with gallbladder adenocarcinoma treated by cholecystectomy at the Kangbuk Samsung Hospital from 2000 to 2011. CK17 expression was examined by immunohistochemistry and correlated with clinicopathologic prognostic factors. CK17 stained the cytoplasm of tumor cells and immunohistochemical interpretation was possible in 77 cases. Among these, 41 (53.2%) were considered positive using a 5% cutoff determined by a receiver operating characteristic curve (area under the curve=0.656, P=0.021). CK17 expression was associated with poor tumor differentiation (P<0.001), high pT stage (P<0.001), presence of distant metastasis (P=0.036), and low disease-specific survival rate (P<0.001). These results indicate that CK17 can be used as a marker for poor prognosis for gallbladder adenocarcinoma.

The hedgehog signaling plays supportive roles in various aspects of tumorigenesis. Increased expression of the key component, GLI1, has been shown to correlate with poor prognosis in many types of cancers. We aimed to investigate the effect of GLI1 expression in glioblastoma focusing on the nuclear localization. Immunohistochemistry for GLI1, GLI2, PTCH1, SMO, and SHH were done in 140 glioblastoma tissues, and the staining was graded. For GLI1, nuclear and cytoplasmic expression was separately assessed. No significant correlation was found between clinicopathologic parameters and expression grades of the five proteins. Low nuclear GLI1 expression was associated with a worse progression-free survival while overall survival was not significantly affected. In contrast, cytoplasmic GLI1 expression did not have a prognostic effect. PTCH1 expression correlated with nuclear GLI1 expression without exerting a significant prognostic effect. Analysis of the TCGA-glioblastoma dataset revealed that low GLI1 mRNA level also correlated with a poor prognosis for both overall and progression-free survival. The adverse effect of low nuclear GLI1 expression in glioblastomas is in contrast with the negative prognostic effect of high GLI1 expression reported in non-cranial malignancies. The relative impact of hedgehog signaling among other oncogenic pathways in the brain may be responsible for the difference. The different implication of GLI1 expression in glioblastomas needs to be considered in studies of hedgehog signaling-targeted therapy.

Background: Smad4 and GATA3 proteins are known prognostic markers in various cancers. Smad4 is a mediator linked to both tumour suppression and progression. GATA3 is a regulator of development and morphogenesis of the mammary gland. We assessed and compared the predictive performance of Smad4 and GATA3 for clinical outcomes in patients with breast cancer., Methods: The combined expression pattern based on Smad4+/- and GATA3+/- was evaluated by immunostaining using breast cancer tissue microarray, and the relationships between protein expression and clinicopathological variables were analysed., Results: Smad4 expression was only associated with an ill-defined tumour border, whereas GATA3 was associated with several good prognostic factors. On analysis of combined markers, there was a significant difference in the expression of fascin (an important factor for cancer invasiveness) between the Smad4+/GATA3- and Smad4-/GATA3+ groups. Smad4+/GATA3- was correlated with worse clinicopathological parameters, relapse-free survival (RFS), and overall survival (OS), compared to Smad4-/GATA3+., Conclusion: Combined markers of Smad4/GATA3 showed a superior performance compared to single markers for predicting RFS and OS in patients with breast cancer., (© 2017 S. Karger AG, Basel.)

Aim: We investigated the feasibility of dynamin 2 as a potential treatment target in cervical cancer cells., Materials and Methods: We performed tissue microarray for dynamin 2 expression in 208 patients with early cervical cancer and in vitro in HeLa cells with dynamin 2 inhibitors MiTMAB, OcTMAB, Dynasore, and DD-6., Results: Tumor size greater than 2 cm or tumor invasion of more than half of the entire cervix was associated with expression of dynamin 2 compared to no expression (p=0.013, and p=0.045, respectively). All dynamin 2 inhibitors significantly reduced proliferation, increased apoptotic activity, and reduced matrix metallopeptidase 9 expression in HeLa cells. Dynasore and DD-6 reduced migration of HeLa cells on laminin 1-coated plates and DD-6 most strongly reduced migration performance on fibronectin-coated plates., Conclusion: Targeting dynamin 2 may be a promising new approach for the treatment of cervical cancer., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Purpose: Progranulin (PGRN), characterized as an autocrine growth and survival factor, is known to stimulate the proliferation and survival of several cancer cell types. However, little is known about the prognostic role of PGRN in advanced biliary tract cancers (BTCs)., Methods: A retrospective analysis was performed on patients with advanced BTC who received palliative chemotherapy between July 2004 and November 2014. PGRN expression was immunohistochemically evaluated according to staining intensity of tumor and peritumoral cells., Results: A total of 80 patients (39 intrahepatic, 26 extrahepatic, and 18 gallbladder tumors) were analyzed. The median age was 64 years (range 31-79), and 48 patients (60 %) were male. Thirty-five patients (44 %) had high tumor PGRN expression (PGRN positive), and there was a trend of poorer response to chemotherapy in patients with PGRN-positive tumor in terms of overall response rate (7 vs. 18 %). With a median follow-up duration of 17.7 months (range 4.9-35.1), PGRN-positive patients had worse progression-free survival (PFS) with a median of 2.7 months compared to 5.0 months for PGRN-negative patients (P = 0.023). After adjusting for possible confounding factors including sex, age, performance status, disease status, and chemotherapy agent, multivariate analysis showed that PGRN-positive tumor was a prognostic factor independently associated with poor PFS (hazard ratio 1.69, 95 % CI 1.02-2.81; P = 0.044)., Conclusion: PGRN overexpression was significantly associated with poor PFS in advanced BTCs. PGRN expression by IHC analysis might help predict treatment outcomes and provide a new target for molecular therapy.

Background & Aims: This study is designed to investigate the expression of survivin and p53 in human rectal cancer tissues and analyze associations between expression and clinical outcomes in terms of disease recurrence and survival duration., Results: During follow-up (median 119.0, range 6.6 to 161.3 months), tumor recurrence was detected in 50 patients (43.1%), and local recurrence developed as a first failure site in 13 patients (11.2%). Positive immunostaining of nuclear and cytoplasmic survivin was observed in about one quarter of patients, and about half of all patients had positive staining for p53. Both survivin and p53 were significant prognostic factors of disease-free survival in the univariate analyses, but only survivin remained a significant prognostic factor in the multivariate analysis., Methods: We performed a retrospective study with 116 locally advanced rectal cancer patients who underwent total mesorectal excision (TME) followed by postoperative concurrent chemo-radiation therapy (CCRT). Immunohistochemical staining was conducted using antibodies for survivin or p53, and their expression was analyzed using an individual score that combined the percentage of positive cells and staining intensity., Conclusions: Overexpression of nuclear and cytoplasmic survivin in locally advanced rectal cancer patients was associated with a higher recurrence rate in rectal cancer patients treated with TME followed by postoperative CCRT., Competing Interests: The authors declared that they have no conflicts of interest related to this work.

Background: Previous studies have demonstrated aberrant Brahma-related gene 1 (BRG1) expression in various tumor types. Increased BRG1 expression has recently been shown to correlate with aggressive oncogenic behavior in many different types of human cancer. However, the role of BRG1 in breast cancer development and progression is not fully understood., Materials and Methods: We evaluated BRG1 expression in 224 patients with invasive ductal carcinoma (IDC) of the breast using tissue microarray samples and immunohistochemistry. We also investigated whether BRG1 expression status is associated with clinicopathological characteristics and outcomes of patients with IDC., Results: Among the 224 patients with IDC, 37.5% (84/224) exhibited high BRG1 expression. IDC exhibited significantly higher BRG1 expression compared to ductal carcinoma in situ (p=0.009) and normal breast tissue (p=0.005). High BRG1 expression in IDC significantly correlated with higher histological grade (p=0.035) and presence of distant metastasis (p=0.002). Furthermore, high BRG1 expression was an independent factor for predicting distant metastasis (relative risk=4.079; p=0.007). In addition, high BRG1 expression predicted shorter overall (p=0.011) and recurrence-free (p=0.003) survival in patients with IDC. In particular, BRG1 had a significant prognostic value in predicting recurrence-free survival of patients with IDC with lymph node metastasis or stage III disease., Conclusion: BRG1 is involved in the progression and metastasis of breast cancer and can serve as a novel biomarker predictive of distant metastasis and patient outcomes., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

The biology of breast cancer brain metastasis (BCBM) is poorly understood. We aimed to explore genes that are implicated in the process of brain metastasis of primary breast cancer (BC). NanoString nCounter Analysis covering 252 target genes was used for comparison of gene expression levels between 20 primary BCs that relapsed to brain and 41 BCBM samples. PAM50-based intrinsic subtypes such as HER2-enriched and basal-like were clearly over-represented in BCBM. A panel of 22 genes was found to be significantly differentially expressed between primary BC and BCBM. Five of these genes, CXCL12, MMP2, MMP11, VCAM1, and MME, which have previously been associated with tumor progression, angiogenesis, and metastasis, clearly discriminated between primary BC and BCBM. Notably, the five genes were significantly upregulated in primary BC compared to BCBM. Conversely, SOX2 and OLIG2 genes were upregulated in BCBM. These genes may participate in metastatic colonization but not in primary tumor development. Among patient-matched paired samples (n = 17), a PAM50 molecular subtype conversion was observed in eight cases (47.1%), with a trend toward unfavorable subtypes in patients with the distinct gene expression. Our findings, although not conclusive, reveal differentially expressed genes that might mediate the brain metastasis process.

Cell cycle regulatory proteins including p16, p27, and p53 are well studied in various cancers. However, their single or concurrent roles related with the clinicopathological parameters are not clearly recognized. We analyzed the expression of p16, p27, and p53 cell cycle regulatory proteins in papillary thyroid carcinoma (PTC). To determine the prognostic significance of cell cycle regulatory proteins, 107 PTCs were examined. We analyzed the individual expression of p16, p27, and p53 and their concurrent expressions, with the relationship to various clinicopathological parameters including differentiation from benign lesions. High expression of p16 and p53 and low expression of p27 were related with the distinguishing of PTC from benign lesions. In addition, normal thyroidal tissue showed higher p27 expression than nodular hyperplasia. In relation to extrathyroidal extension (ETE), the low expression of p27 was related with the presence of ETE. The low expression of p27 and high expression of p16 and p53 may affect the development of PTC. In addition, low p27 expression is related with the existence of ETE.

Background: Triple-negative breast cancer (TNBC) is known for aggressive biologic features and poor prognosis. Epidermal growth factor receptor (EGFR) overexpression in TNBC indicates poor prognosis. However, there is no previous study of the relationship between expression of the entire human epidermal growth factor receptor (HER) family genes and patient prognosis in TNBC. Accordingly, we investigated the expression profiles of HER family genes in patients with TNBC to determine the prognostic value and clinical implications of HER family expression., Methods: We used the nCounter expression assay (NanoString®) to measure the expression of EGFR, erb-B2 receptor tyrosine kinase 2 (ERBB2), ERBB3, ERBB4, and estrogen receptor 1 (ESR1) genes using mRNA extracted from paraffin-embedded tumor tissues from 203 patients diagnosed with TNBC. Our data were validated using a separate cohort of 84 TNBC patients., Results: A total of 203 TNBC patients who received adjuvant chemotherapy after curative surgery from 2000 to 2004 formed the training set. The 84 TNBC patients in the validation consort were selected from breast cancer patients who received curative surgery since 2005 to 2010. Analysis of the expression profiles of the HER family genes in TNBC tissue specimens revealed that increased expression of ERBB4 was associated with poor prognosis according to survival analysis (5-year distant relapse free survival [5Y DRFS], low vs. high expression [cut-off: median]: 90.1% vs. 80.2%; p = 0.022). This trend was also observed in the validation set of TNBC patients (5Y DRFS, low vs. high: 69.4% vs. 44.7%; p = 0.053). In a multivariate Cox regression model, ERBB4 expression was identified as a indicator of long-term prognosis in patients with TNBC., Conclusions: The expression profile of ERBB4, a member of the HER family, might serve as a prognostic marker in patients with TNBC.

Although breast cancer is the second most common cause of brain metastasis with a notable increase of incidence, genes that mediate breast cancer brain metastasis (BCBM) are not fully understood. To study the molecular nature of brain metastasis, we performed gene expression profiling of brain metastasis and matched primary breast cancer (BC). We used the Ion AmpliSeq Cancer Panel v2 covering 2,855 mutations from 50 cancer genes to analyze 18 primary BC and 42 BCBM including 15 matched pairs. The most common BCBM subtypes were triple-negative (42.9%) and basal-like (36.6%). In a total of 42 BCBM samples, 32 (76.2%) harbored at least one mutation (median 1, range 0-7 mutations). Frequently detected somatic mutations included TP53 (59.5%), MLH1 (14.3%), PIK3CA (14.3%), and KIT (7.1%). We compared BCBM with patient-matched primary BC specimens. There were no significant differences in mutation profiles between the two groups. Notably, gene expression in BCBM such as TP53, PIK3CA, KIT, MLH1, and RB1 also seemed to be present in primary breast cancers. The TP53 mutation frequency was higher in BCBM than in primary BC (59.5% vs 38.9%, respectively). In conclusion, we found actionable gene alterations in BCBM that were maintained in primary BC. Further studies with functional testing and a delineation of the role of these genes in specific steps of the metastatic process should lead to a better understanding of the biology of metastasis and its susceptibility to treatment.

Background: Wnt5a, a non-canonical Wnt ligand, has been shown to play tumor-promoting or tumor-suppressive roles in different neoplasms. Increased Wnt5a expression and Wnt5a-dependent invasive activity that is mediated by one of its receptors, Ryk, have been reported in glioblastomas., Methods: We investigated the protein expression of Wnt5a, its receptors Ryk and Ror2, and the canonical Wnt pathway marker β-catenin in 186 cases of glioblastoma and its variants. Associations with clinicopathological and molecular variables and prognosis were analyzed., Results: All glioblastoma cases expressed Wnt5a, Ryk and Ror2 with a different grade. The expression of both Ryk and Ror2 correlated with that of Wnt5a in glioblastomas. The expression of β-catenin did not correlate with any of Wnt5a, Ryk or Ror2. Wnt5a expression was significantly different among subgroups of the glioblastoma. However, none of Wnt5a, Ryk or Ror2 had a prognostic impact on glioblastoma. For β-catenin, a shorter progression-free survival was noted in the glioblastoma with oligodendroglioma component (GBMO) subgroup., Conclusions: Our results corroborated previous findings of Ryk-mediated Wnt5a effect, and suggested a role for Ror2 in the Wnt5a machinery in glioblastoma., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

This study was designed to investigate whether proton pump inhibitors (PPI, V-ATPase blocker) could increase the effect of cytotoxic agents in chemoresistant epithelial ovarian cancer (EOC). Expression of V-ATPase protein was evaluated in patients with EOC using immunohistochemistry, and patient survival was compared based on expression of V-ATPase mRNA from a TCGA data set. In vitro, EOC cell lines were treated with chemotherapeutic agents with or without V-ATPase siRNA or PPI (omeprazole) pretreatment. Cell survival and apoptosis was assessed using MTT assay and ELISA, respectively. In vivo experiments were performed to confirm the synergistic effect with omeprazole and paclitaxel on tumor growth in orthotopic and patient-derived xenograft (PDX) mouse models. Expression of V-ATPase protein in ovarian cancer tissues was observed in 44 patients (44/59, 74.6%). Higher expression of V-ATPase mRNA was associated with poorer overall survival in TCGA data. Inhibition of V-ATPase by siRNA or omeprazole significantly increased cytotoxicity or apoptosis to paclitaxel in chemoresistant (HeyA8-MDR, SKOV3-TR) and clear cell carcinoma cells (ES-2, RMG-1), but not in chemosensitive cells (HeyA8, SKOV3ip1). Moreover, the combination of omeprazole and paclitaxel significantly decreased the total tumor weight compared with paclitaxel alone in a chemoresistant EOC animal model and a PDX model of clear cell carcinoma. However, this finding was not observed in chemosensitive EOC animal models. These results show that omeprazole pretreatment can increase the effect of chemotherapeutic agents in chemoresistant EOC and clear cell carcinoma via reduction of the acidic tumor microenvironment.

Background: More than half of the patients selected based on KRAS mutation status fail to respond to the treatment with cetuximab in metastatic colorectal cancer (mCRC). We designed a study to identify additional biomarkers that could act as indicators for cetuximab treatment in mCRC., Methods: We investigated 58 tumor samples from wild type KRAS CRC patients treated with cetuximab plus irinotecan (CI). We conducted the genotyping for mutations in either BRAF or PIK3CA and profiled comprehensively the expression of 522 kinase genes., Results: BRAF mutation was detected in 5.1 % (3/58) of patients. All 50 patients showed wild type PIK3CA. Gene expression patterns that categorized patients with or without the disease control to CI were compared by supervised classification analysis. PSKH1, TLK2 and PHKG2 were overexpressed significantly in patients with the disease control to IC. The higher expression value of PSKH1 (r = 0.462, p < 0.001) and TLK2 (r = 0.361, p = 0.005) had the significant correlation to prolonged PFS., Conclusion: The result of this work demonstrated that expression nature of kinase genes such as PSKH1, TLK2 and PHKG2 may be informative to predict the efficacy of CI in wild type KRAS CRC. Mutations in either BRAF or PIK3CA were rare subsets in wild type KRAS CRC.

In women with metastatic breast cancer (MBC), introduction of the anti-HER2 (human epidermal growth factor receptor-2) directed therapies including trastuzumab, pertuzumab, lapatinib, and/or trastuzumab-DM1 has markedly improved overall survival. However, not all cases of HER2-positive breast tumours derive similar benefit from HER2-directed therapy, and a significant number of patients experience disease progression because of primary or acquired resistance to anti-HER2-directed therapies. We integrated genomic and clinicopathological analyses in a cohort of patients with refractory breast cancer to anti-HER2 therapies to identify the molecular basis for clinical heterogeneity. To study the molecular basis underlying refractory MBC, we obtained 36 MBC tumours tissues and used next-generation sequencing to investigate the mutational and transcriptional profiles of 83 genes. We focused on HER2 mutational sites and HER2 pathways to identify the roles of HER2 mutations and the HER2 pathway in the refractoriness to anti-HER2 therapies. Analysis using massively parallel sequencing platform, CancerSCAN™, revealed that HER2 mutations were found in six of 36 patients (16.7%). One patient was ER (estrogen receptor)-positive and HER2-negative and the other five HER2 mutated patients were HER2-positive and HR (hormone receptor)-negative. Most importantly, four of these five patients did not show any durable clinical response to HER2-directed therapies. The HER2 pathway score obtained through transcriptional analyses identified that Growth Receptor Biding protein 2 (GRB2) was the most significantly down regulated gene in the HER2 mutated samples. Detection of HER2 mutations using higher deep DNA sequencing may identify a predictive biomarker of resistance to HER2-directed therapy. Functional validation is warranted.

Background: In this study, we established patient-derived tumor cell (PDC) models using tissues collected from patients with metastatic cancer and assessed whether these models could be used as a tool for genome-based cancer treatment., Methods: PDCs were isolated and cultured from malignant effusions including ascites and pleural fluid. Pathological examination, immunohistochemical analysis, and genomic profiling were performed to compare the histological and genomic features of primary tumors, PDCs. An exploratory gene expression profiling assay was performed to further characterize PDCs., Results: From January 2012 to May 2013, 176 samples from patients with metastatic cancer were collected. PDC models were successfully established in 130 (73.6%) samples. The median time from specimen collection to passage 1 (P1) was 3 weeks (range, 0.5-4 weeks), while that from P1 to P2 was 2.5 weeks (range, 0.5-5 weeks). Sixteen paired samples of genomic alterations were highly concordant between each primary tumor and progeny PDCs, with an average variant allele frequency (VAF) correlation of 0.878. We compared genomic profiles of the primary tumor (P0), P1 cells, P2 cells, and patient-derived xenografts (PDXs) derived from P2 cells and found that three samples (P0, P1, and P2 cells) were highly correlated (0.99-1.00). Moreover, PDXs showed more than 100 variants, with correlations of only 0.6-0.8 for the other samples. Drug responses of PDCs were reflective of the clinical response to targeted agents in selected patient PDC lines., Conclusion(s): Our results provided evidence that our PDC model was a promising model for preclinical experiments and closely resembled the patient tumor genome and clinical response.

Neoadjuvant chemotherapy (NAC) has the added advantage of increasing breast conservation rates with equivalent survival outcomes compared with adjuvant chemotherapy. A subset of breast cancer patients who received NAC experienced early failure (EF) during the course of therapy or within a short period after curative breast surgery. In contrast, patients with pathological complete response (pCR) were reported to have markedly favorable outcomes. This study was performed to identify actionable mutation(s) and to explain refractoriness and responsiveness to NAC. Included in this analysis were 76 patients among 397 with locally advanced breast cancer for whom a preoperative fresh-frozen paraffin-embedded tumor block was available for next-generation sequencing using AmpliSeq. The incidence of missense mutations in KRAS was much higher in patients with EF than in other groups (p < 0.01). In contrast, polymorphisms of the cMET gene were found in patients with pCR exclusively (p < 0.01).

Purpose: Anaplastic lymphoma kinase (ALK) rearrangement has been detected in colorectal carcinoma (CRC) using advanced molecular diagnostics tests including exon scanning, fluorescence in situ hybridization (FISH), and next generation sequencing (NGS). We investigated if immunohistochemistry (IHC) can be used to detect ALK rearrangement in gastrointestinal malignancies., Experimental Designs: Tissue microarrays (TMAs) from consecutive gastric carcinoma (GC) and CRC patients who underwent surgical resection at Samsung Medical Center, Seoul, Korea were screened by IHC using ALK monoclonal antibody 5A4. IHC positive cases were confirmed by FISH, nCounter assays, and NGS-based comprehensive genomic profiling (CGP). ALK IHC was further applied to CRC patients enrolled in a pathway-directed therapeutic trial., Results: Four hundred thirty-two GC and 172 CRC cases were screened by IHC. No GC sample was ALK IHC positive. One CRC (0.6%) was ALK IHC positive (3+) that was confirmed by ALK FISH and a novel CAD-ALK (C35; A20) fusion variant that resulted from a paracentric inversion event inv(2)(p22-21p23) was identified by CGP. One out of 50 CRC patients enrolled in a pathway-directed therapeutic trial was ALK IHC positive (3+) confirmed by ALK FISH and found to harbor the EML4-ALK (E21, A20) fusion variant by CGP. Growth of a tumor cell line derived from this EML4-ALK CRC patient was inhibited by ALK inhibitors crizotinib and entrectinib., Conclusions: ALK IHC is a viable screening strategy for identifying ALK rearrangement in CRC. ALK rearrangement is a potential actionable driver mutation in CRC based on survival inhibition of patient tumor-derived cell line by potent ALK inhibitors.

Sphingosine kinase 1 (SK1) is over-expressed in multiple types of human cancer. SK1 has growth-promoting effects and has been proposed as a potential therapeutic target. We investigated the therapeutic effects of SK1 inhibition in epithelial ovarian carcinoma (EOC). SK1 siRNA or inhibitors were tested in EOC cell lines, including A2780, SKOV3ip1, A2780-CP20, SKOV3-TR, ES2 and RMG2. Cells were treated with SK inhibitor or FTY720, and cell proliferation, apoptosis, angiogenesis and invasion were examined by MTT, FACS, ELISA and wound-healing assays, respectively. In vivo experiments were performed to test the effects of FTY720 on tumor growth in orthotopic mouse xenografts of EOC cell lines A2780 or SKOV3ip1 and a patient-derived xenograft (PDX) model of clear cell ovarian carcinoma (CCC). Blocking SK1 with siRNA or inhibitors significantly reduced proliferation, angiogenesis and invasion, and increased apoptosis in chemosensitive (A2780 and SKOV3ip1) and chemoresistant (A2780-CP20, SKOV3-TR, ES2 and RMG2) EOC cells. SK1 inhibitors also decreased the intracellular enzymatic activity of SK1. Furthermore, FTY720 treatment significantly decreased the in vivo tumor weight in xenograft models of established cell lines (A2780 and SKOV3ip1) and a PDX model for CCC compared to control (p < 0.05). These results support therapeutic targeting of SK1 as a potential new strategy for EOC., (© 2014 UICC.)

Objectives: The aim of this study was to investigate microRNAs related to postoperative prognosis of pancreatic cancer by applying microarray analysis to stored surgical specimens., Methods: We retrospectively analyzed the relationship between microRNA expression and postoperative survival of pancreatic cancer patients in Samsung Medical Center. After the correlation of microRNA expression between paraffin-embedded and fresh-frozen specimens was confirmed, we applied NanoString nCounter system to formalin fixed paraffin-embedded surgical specimens between 1995 September and 2010 June., Results: Paired fresh and paraffin-embedded samples from 9 patients showed a high correlation (correlation coefficient > 0.91). Among 734 microRNAs, 7 microRNAs were found to be related to postoperative overall survival of 221 pancreatic cancer patients: hsa-miR-99a, hsa-miR-150, hsa-miR-194, hsa-miR-375, hsa-miR-664, hsa-miR-342-3p, and hsa-miR-487b (P < 0.01). The prognostic microRNA signature was consistent after adjustment of clinical parameters: resection status, stages, age, sex, adjuvant treatment, pathological differentiation and postoperative carbohydrate antigen 19-9 levels., Conclusions: We found a prediction model for postoperative prognosis of pancreatic cancer using 7 microRNA expression patterns from archived formalin-fixed paraffin-embedded surgical specimens. The methods for microRNA detection and new microRNAs detected in this study provide new insights for new research of surgically resected pancreatic cancer samples and microRNAs.

Viral oncogenes and host immunosenescence have been suggested as causes of Epstein-Barr virus-positive diffuse large B-cell lymphoma (EBV + DLBCL) of the elderly. To investigate the molecular genetic basis of immune evasion and tumor outgrowth, we analyzed copy number alterations (CNAs) and gene expression profiles in EBV + DLBCL samples compared with EBV - DLBCL. There were relatively few genomic alterations in EBV + DLBCL compared with those detected in EBV-negative DLBCL. The most frequent CNAs (>30%) in EBV + DLBCLs were gains at 1q23.2-23.3, 1q23.3, 1q32.1, 5p15.3, 8q22.3, 8q24.1-24.2, and 9p24.1; losses at 6q27, 7q11.2, and 7q36.2-36.3 were also recurrent. A gene expression profile analysis identified the host immune response as a key molecular signature in EBV + DLBCL. Antiviral response genes, proinflammatory cytokines, and chemokines associated with the innate immune response were overexpressed, indicating the presence of a virusinduced inflammatory microenvironment. Genes associated with the B-cell receptor signaling pathway were downregulated. An integrated analysis indicated that SLAMF1 and PDL2 were key targets of the gains detected at 1q23.2-23.3 and 9p24.1. The chromosomal gain at 9p24.1 was associated with poor overall survival. Taken together, our results led to the identification of recurrent copy number alterations and distinct gene expression associated with the host immune response in EBV + DLBCL. We suggest that the upregulation of PDL2 on 9p24.1 promotes immune evasion and is associated with poor prognosis in EBV + DLBCL., (© 2015 Wiley Periodicals, Inc.)

Introduction: Molecular targets are emerging rapidly and the development of clinical tests that simultaneously screen for multiple targets has become especially important. We assessed the gene expression levels of three known targets in advanced gastric cancer, epidermal growth factor receptor (EGFR), human epidermal growth factor 2 (HER2), and N-methyl-N-nitrosoguanidine human osteosarcoma transforming gene (MET), using the nCounter® assay (NanoString Technologies, Seattle, WA, USA) and compared these results with protein overexpression, detected by immunohistochemistry, to evaluate the performance of this new technology., Methods: We investigated 42 formalin-fixed, paraffin-embedded tumor samples from patients with gastric cancer. A NanoString-based assay containing a 522 kinase gene panel was investigated. We analyzed the correlations between immunohistochemical findings and kinase gene expression levels of EGFR, HER2 and MET to validate this assay., Results: EGFR, HER2, and MET overexpression were observed in 7 (16.6 %), 5 (11.9 %), and 3 (7.1 %) cases, respectively. For EGFR, HER2, and MET, the concordance rates between the NanoString-based assay results and the immunohistochemistry methods were 83.3, 97.6, and 100 %, respectively. Relative to immunohistochemistry findings, the NanoString-based assay sensitivities and specificities were 85.7 and 82.8 % for EGFR, 100 and 97.2 % for HER2, and 100 and 100 % for MET, respectively., Conclusions: We found a high concordance between immunohistochemistry- and nCounter-based assessments of EGFR, HER2, and MET in advanced gastric cancer. Judged against immunohistochemistry results, the NanoString assay had high sensitivities and high specificities. These results suggest that the nCounter assay provides a reliable, high-throughput assay to simultaneously screen for the overexpression of several target proteins.

Purpose: Efficacy of gefitinib therapy strongly depends on epidermal growth factor receptor (EGFR) mutation status in patients with non-small cell lung cancer (NSCLC). However, cumulative data from many clinical studies demonstrated that some patients with wild-type (WT) EGFR also responded to gefitinib with durable disease control rate (DCR). The aim of this trial was to evaluate the efficacy and toxicity of gefitinib in NSCLC patients with WT EGFR who failed previous chemotherapy., Patients and Methods: Patients with advanced or recurrent NSCLC whose tumors have WT EGFR were eligible. Gefitinib (250 mg/day) was administered until disease progression or unacceptable toxicity occurred. The primary end point was DCR at 8 weeks., Results: A total of 85 patients (53 men and 32 women; median age, 60 years; range 30-86) were enrolled between October 2010 and May 2013. Seventy-four patients (87.1 %) had adenocarcinoma. Forty-two patients (49.4 %) were treated with gefitinib as second-line chemotherapy. Eleven patients showed partial response, and 21 had stable disease. Thus, objective response rate was 12.9 %, and DCR at 8 weeks was 37.6 %. The median progression-free survival (PFS) and overall survival were 1.9 and 10.9 months, respectively. Skin rash was the most common side effect. It is of note that patients with skin rash of any grade had improved PFS with gefitinib as compared with patients experiencing no skin rash (median PFS: 3.0 vs. 1.7 months, P = 0.004). One patient developed interstitial lung disease (grade 2). Of 11 gefitinib responders, 6 patients were identified as having tumor with activating EGFR mutation by peptide nucleic acid (PNA)-mediated PCR clamping method. Regarding the outcomes of the 79 patients, excluding 6 positive mutations, the response rate was 6.3 %, and DCR at 8 weeks was 31.8 %., Conclusion: Small proportion of NSCLC patients with the WT EGFR benefits with gefitinib. Optimized diagnosis through more sensitive bioassay could have major consequences in terms of the selection of candidate for EGFR TKI in patients with WT EGFR by direct sequencing.